1.1 Field of the Invention
The present invention relates to the use of fluorogenic or chromogenic dyes as reporter molecules for detecting cell entry by a specific molecule. The present invention has application to cellular assays, including high throughput assays that utilize fluorogenic or chromogenic reporters to detect transportation of a molecule across a cell membrane.
1.2 The Related Art
Numerous fluorescent and fluorogenic dyes have been used as reporters in fluorodetection assays. In particular, numerous fluorescein derivatives have been reported which possess functional groups that are suitable for reacting with other molecules and have been used as tracers in analytical applications ranging from the probing of cell functions to the monitoring of the level of one or more drugs in physiological test samples. See, e.g., C. Dive, et al., Mol. Cell. Probes 2:31 (1988); Graber, et al., Anal. Biochem. 156:202 (1986); P. J. Brynes, et al., U.S. Pat. No. 4,869,132 and N. Y. Wang, et al., EP 264797. Examples of analytical applications wherein such compounds are used include by way of example fluorescent polarization immunoassays (FPIAs) for use in commercially available instruments such as the Abbott ADx and Abbott TDx instruments (both available from Abbott Labs, Abbott Park, Ill.). Examples of such derivatives include 5- and 6-amino fluorescein (M. T. Ship Chandler, et al., Anal. Biochem. 162:89 (1987); Mattingly, U.S. Pat. No. 5,573,904 (1996) and U.S. Pat. No. 5,756,771 (1996), and Ghoshal, et al, U.S. Pat. No. 5,986,094 (1999)).
Fluorescein dyes particularly have been used to detect cell entry. For example, fluorescein 1 has been employed especially to detect cell entry by peptides. Additionally, carboxyfluorescein diacetate 2 and its derivatives find known application in staining liver cells because upon entry a cellular esterase cleaves the ester moiety resulting in the generation of fluorescein, which is highly fluorescent. Monobromobimane 3, which does not fluoresce until reacted with a thiol, has also been used to detect cell entry. In particular, this compound does not fluoresce until it contacts the cell cytoplasm and interacts with glutathione, a tripeptide thiol. Thus, 3 can be used to detect cell entry or intracellular thiol levels based on an increase in fluorescence relative to the extracellular environment, which typically contains little or no free thiol. Further, electron-deficient heterocycle-substituted fluorescent dyes have been used as fluorimetric reporter molecules (see, e.g., U.S. Pat. No. 6,221,604).

However, while fluorescein derivatives have been widely used as reporter molecules to detect cell entry, known fluorescein derivatives suffer from significant disadvantages. For example, the use of known fluorescein dyes to detect cell entry requires tedious separation of intracellular fluorescence from extracellular fluorescence; and, therefore, the use of fluorescein dyes to detect cell entry are not amenable to high-throughput assays. Moreover, carboxyfluorescein diacetate 1 suffers from a short half-life, which has been attributed to the hydrolysis of its ester moieties at physiological pH.
Certain peptides are recognized to possess the ability to enter cells as well as to transport attached molecules into cells. Examples of such peptides include those derived from HIV tat protein, lysine polymers, Antennapedia homeodomain, and Arg 9 among others. (See e.g., U.S. Pat. No. 5,804,604 by Frank, et al., relating to the use of HIV tat protein derivatives to facilitate intracellular delivery of cargo molecules; WO 98/52614, which discloses the use of arginine polymers containing guanidino or amidino side chains to facilitate cell entry; WO 79/00515, which discloses the use of high molecular weight lysine polymers to facilitate cell entry of target molecules; and WO 94/04686 (1984) and Fawell, et al., Proc. Natl. Acad, Sci., USA 91:664–668 (1994), each of which discloses the use of peptides derived from HIV tat to promote the transport of molecules across cell membranes). Also, certain peptoid sequences have been demonstrated to facilitate intracellular transport (Wender, et al., Proc. Natl. Acad. Sci., USA 97:13003–13008 (2000)). Arginine oligomers have also been reported to deliver topically a cyclic peptide drug, cyclosporin A, into cells to inhibit inflammation (Rothbard, et al., Nature Med. 6:1253–7 (2000)).
Physicochemical methods to facilitate the delivery of macromolecules into cells have been developed. Such methods include, by way of example, electroporation, membrane fusion with liposomes, calcium-phosphate-DNA precipitation, DEAE-dextran-mediated transfection, infection with modified nucleic acids and direct microinjection into cells.
However, despite foregoing descriptions of fluorogenic compounds and carrier molecules, the identification of new fluorogenic molecules that are effectively transported into cells and more efficient methods for identifying such molecules would be beneficial.